Andrzej Pokrywka

GENES IN SPORT AND DOPING

Genes control biological processes such as muscle production of energy, mitochondria biogenesis, bone formation, erythropoiesis, angiogenesis, vasodilation, neurogenesis, etc. DNA profiling for athletes reveals genetic variations that may be associated with endurance ability, muscle performance and power exercise, tendon susceptibility to injuries and psychological aptitude. Already, over 200 genes relating to physical performance have been identified by several research groups. Athletes’ genotyping is developing as a tool for the formulation of personalized training and nutritional programmes to optimize sport training as well as for the prediction of exercise-related injuries. On the other hand, development of molecular technology and gene therapy creates a risk of non-therapeutic use of cells, genes and genetic elements to improve athletic performance. Therefore, the World Anti-Doping Agency decided to include prohibition of gene doping within their World Anti-Doping Code in 2003. In this review article, we will provide a current overview of genes for use in athletes’ genotyping and gene doping possibilities, including their development and detection techniques.

Overrepresentation of the COL3A1 AA genotype in Polish skiers with anterior cruciate ligament injury

Although various intrinsic and extrinsic risk factors for anterior cruciate ligament (ACL) rupture have been identified, the exact aetiology of the injury is not yet fully understood. Type III collagen is an important factor in the repair of connective tissue, and certain gene polymorphisms may impair the tensile strength. The aim of this study was to examine the association of the COL3A1 rs1800255 polymorphism with ACL rupture in Polish male recreational skiers. A total of 321 male Polish recreational skiers were recruited for this study; 138 had surgically diagnosed primary ACL ruptures (ACL-injured group) and 183 were apparently healthy male skiers (control group – CON) who had no self-reported history of ligament or tendon injury. Both groups had a comparable level of exposure to ACL injury. Genomic DNA was extracted from the oral epithelial cells. All samples were genotyped on a real-time polymerase chain reaction instrument. The genotype distribution in the ACL-injured group was significantly different than in CON (respectively: AA=10.1 vs 2.2%, AG=22.5 vs 36.1, GG=67.4 vs 61.8%; p=0.0087). The AA vs AG+GG genotype of COL3A1 (odds ratio (OR)=5.05; 95% confidence interval (CI), 1.62-15.71, p=0.003) was significantly overrepresented in the ACL-injured group compared with CON. The frequency of the A allele was higher in the ACL-injured group (21.4%) compared with CON (20.2%), but the difference was not statistically significant (p=0.72). This study revealed an association between the COL3A1 rs1800255 polymorphism and ACL ruptures in Polish skiers.

Insights into Supplements with Tribulus Terrestris used by Athletes.

Abstract Herbal and nutritional supplements are more and more popular in the western population. One of them is an extract of an exotic plant, named Tribulus terrestris (TT). TT is a component of several supplements that are available over-the-counter and widely recommended, generally as enhancers of human vitality. TT is touted as a testosterone booster and remedy for impaired erectile function; therefore, it is targeted at physically active men, including male athletes. Based on the scientific literature describing the results of clinical trials, this review attempted to verify information on marketing TT with particular reference to the needs of athletes. It was found that there are few reliable data on the usefulness of TT in competitive sport. In humans, a TT extract used alone without additional components does not improve androgenic status or physical performance among athletes. The results of a few studies have showed that the combination of TT with other pharmacological components increases testosterone levels, but it was not discovered which components of the mixture contributed to that effect. TT contains several organic compounds including alkaloids and steroidal glycosides, of which pharmacological action in humans is not completely explained. One anti-doping study reported an incident with a TT supplement contaminated by a banned steroid. Toxicological studies regarding TT have been carried out on animals only, however, one accidental poisoning of a man was described. The Australian Institute of Sport does not recommend athletes’ usage of TT. So far, the published data concerning TT do not provide strong evidence for either usefulness or safe usage in sport.

Detection of β-methylphenethylamine, a novel doping substance, by means of UPLC/MS/MS

Novel substances of expected doping activity are constantly introduced to the market. β-Methylphenethylamine (BMPEA) is classified as a doping agent by the World Anti-Doping Agency as it is a positional isomer of amphetamine. In this work, the development and application of a simple and rapid analytical procedure that enables discrimination between both isomers is described. The analytes of interest were extracted from urine by a two-step liquid–liquid extraction and then analyzed by UPLC/MS/MS under isocratic conditions. The entire analytical procedure was validated by evaluating its selectivity, discrimination capabilities, carry-over, sensitivity, and influence of matrix effects on its performance. Application of the method resulted in detection of BMPEA in eight anti-doping samples, including the first report of adverse analytical finding regarding its use. Further analysis showed that BMPEA may be eliminated unchanged along with its phase II conjugates, the hydrolysis of which may considerably improve detection capabilities of the method. Omission of the hydrolysis step may therefore, produce false-negative results. Testing laboratories should also carefully examine their LC/MS/MS-based amphetamine and BMPEA findings as both isomers fragment yielding comparable collision-induced dissociation spectra and their insufficient chromatographic separation may result in misidentification. This is of great importance in case of forensic analyses as BMPEA is not controlled by the public law, and its manufacturing, distribution, and use are legal.

THE ASSOCIATION BETWEEN ACE GENE VARIATION AND AEROBIC CAPACITY IN WINTER ENDURANCE DISCIPLINES

The aim of the study was to examine the possible relationship between I/D polymorphism of ACE gene and selected indices of aerobic capacity among male and female athletes practising winter endurance sports. Sixty-six well-trained athletes (female n = 26, male n = 40), aged 18.4 ± 2.8 years, representing winter endurance sports (cross-country skiing, n = 48; biathlon, n = 8; Nordic combined, n = 10) participated in the study. Genotyping for ACE I/D polymorphism was performed using polymerase chain reaction. Maximal oxygen consumption (VO2max), maximal running velocity (Vmax) and running velocity at anaerobic threshold (VAT4) were determined in an incremental test to volitional exhaustion on a motorized treadmill. The ACE genotype had no significant effect on absolute VO2max, relative VO2max (divided by body mass or fat free body mass), VAT4 or Vmax. No interaction effect of gender x ACE genotype was found for each of the examined aerobic capacity indices. ACE gene variation was not found to be a determinant of aerobic capacity in either female or male Polish, well-trained endurance athletes participating in winter sports.

INGESTION OF DESIGNER SUPPLEMENTS PRODUCED POSITIVE DOPING CASES UNEXPECTED BY THE ATHLETES

Since the end of the last century, the sport community has been grappling with contamination and faking of dietary or nutritional supplements. Publication of the results of many studies on this problem has resulted in educational and informative actions on risk connected with consumption of unchecked products. As compared to the past, nowadays athletes have become incomparably more aware of the potential risk of a positive result in anti-doping tests as a consequence of consumption of contaminated dietary supplements. However, this problem is still topical, which is confirmed by regular reports, presented e.g. during the Cologne Workshops on Dope Analysis held annually in Germany. This paper presents a few doping cases caused by the use of supplements with doping substances by athletes, noted by two WADA accredited laboratories (Cologne and Warsaw). Still more education on the health and doping risks of dietary supplement products seems to be necessary for the protection of both athletes and the general public.

Some aspects concerning modifications of the list of prohibited substances and methods in sport.

In 1967 the International Olympic Committee (IOC) founded Medical Commission to organize and supervise fight against doping. At that time, the Commission published the first list of substances prohibited for use in sport to meet the need of anti-doping testing at the 1968 Olympic Games. The Prohibited List included stimulants, sympathomimetic amines, narcotics (narcotic analgesics), antidepressants and tranquilizers. For years the list was expanding and underwent modifications, mainly prior to successive Olympic Games. Starting from 1 January 2004, the World Anti-Doping Agency (WADA) has assumed the role of the main coordinator in fight against doping. WADA significantly modified the list of prohibited substances and methods (the Prohibited List). These modifications initiated changes, whose effects can be observed in three main areas of sport and anti-doping i.e. in: athletes, doping control laboratories, and sport entourage. In Poland, the removal some substances from the List or the addition other compounds to the basic List caused an increase of usage of pseudoephedrine and caffeine by athletes and a decrease of number of positive doping cases with cannabinoids and glucocorticosteroids. The annual modification of the Prohibited List by WADA and subsequent introduction of new examples of prohibited substances strengthened the world anti-doping system. Considering the open character of the list a regular update would be expected, especially indicating prohibited or permitted status of new substances and drugs. It would be advisable to publish, on the WADA website, some additional information regarding those substances which cause the most interpretation problems.

The influence of hypoxic physical activity on cfDNA as a new marker of vascular inflammation

The phenomenon of circulating cell-free DNA (cfDNA) is important for many biomedical disciplines including the field of exercise biochemistry and physiology. It is likely that cfDNA is released into the plasma by apoptosis of endothelial cells and circulating endothelial progenitor cells (EPCs), and/or by NETosis of immune cells induced by strenuous exercise. Increases of cfDNA are described to be a potential hallmark for the overtraining syndrome, and might be related to aseptic vascular inflammation in athletes. Yet, the relevance of systemic inflammation and cfDNA with endothelial dysfunction in athletes still remains unclear. In this review article, we provide a current overview of exercise-induced cfDNA release to the circulation with special emphasis on its relationship with apoptosis and NETosis and the effect of hypoxic physical activity on vascular inflammation in athletes.

Overrepresentation of the ACTN3 XX Genotype in Elite Canoe and Kayak Paddlers

Abstract Orysiak, J, Sitkowski, D, Zmijewski, P, Malczewska-Lenczowska, J, Cieszczyk, P, Zembron-Lacny, A, and Pokrywka, A. Overrepresentation of the ACTN3 XX Genotype in Elite Canoe and Kayak Paddlers. J Strength Cond Res 29(4): 1107–1112, 2015—The aim of the study was to examine the association between the ACTN3 R577X polymorphism in canoe sprint athletes (canoe and kayak paddlers) and their results at 200- or 1000-m distance. Eighty-six European white male athletes divided into 2 groups—successful, who were outstanding at national championships, and nonsuccessful in these competitions—and 354 nonathletic controls were included in this study. The R577X polymorphism of ACTN3 was typed using PCR-RFLP. ACTN3 genotype distribution among all tested athletes and controls was in Hardy-Weinberg equilibrium. The odds ratio (OR) for successful 1000-m athletes harboring the XX genotype compared with sedentary controls was 2.95 (95% confidence interval [CI]: 1.37–6.35), but the OR for nonsuccessful 200-m athletes having the XX genotype compared with controls was 2.64 (95% CI: 1.30–5.36). These results suggest that factors associated with the ACTN3 XX genotype in canoe and kayak paddlers might provide some competitive advantage in performance at 1000 m, but it seems to limit at 200 m. Further studies aimed at development of training strategies based on genetic factors are needed.

The prevalence of trimetazidine use in athletes in Poland: excretion study after oral drug administration

Stimulants, together with anabolic androgenic steroids, are regarded as one of the most popular doping substances in sport. Owing to a great variety of these substances and new designer drugs being introduced to the market, each year the World Anti-Doping Agency (WADA) updates the list of substances and methods prohibited in sport. On 1 January 2014, a new doping agent - trimetazidine (TMZ) - was added to the WADA Prohibited List. TMZ, a substance prohibited in competition, is classified in the S6b Specified Stimulant Group. TMZ is used as a well-known cardiologic drug with confirmed biochemical and clinical activity. According to knowledge of the pharmacology and mechanism of TMZ action, TMZ can be used by athletes to improve physical efficiency, especially in the case of endurance sports. This study presents the phenomena of TMZ use by Polish athletes involved in anti-doping control in the WADA-accredited laboratory in Warsaw (Poland) between 2008 and 2013. Samples were taken from the athletes of such disciplines as cycling, athletics, and triathlon. Moreover, the elimination study of TMZ has been conducted to establish the change of TMZ concentration in urine sample after oral administration of a single or double (during the long-term therapy) dose. TMZ was monitored in urine samples by gas chromatography-mass spectrometry-nitrogen phosphorus detection (GC-MS-NPD).